Therapeutic effects of human mesenchymal stem cell microvesicles in an ex vivo perfused human lung injured with severe E. coli pneumonia

Abstract

Background: We previously reported that microvesicles (MVs) released by human mesenchymal stem cells (MSC) were as effective as the cells themselves in both Escherichia coli lipopolysaccharide and live bacteria-induced acute lung injury (ALI) mice models. However, it remained unclear whether the biological effect of MSC MV can be applied to human ALI.

Methods: In the current study, we tested the therapeutic effects of MSC MVs in a well-established ex vivo perfused human model of bacterial pneumonia. Using human donor lungs not used for transplantation, we instilled E. coli bacteria intrabronchially and, 1 hour later, administered MSC MVs into the perfusate as therapy.

Results: After 6 hours, instillation of E. coli bacteria caused influx of inflammatory cells, which resulted in significant inflammation, lung protein permeability and pulmonary oedema formation. Administration of MSC MV significantly increased alveolar fluid clearance and reduced protein permeability and numerically lowered the bacterial load in the injured alveolus. The beneficial effect on bacterial killing was more pronounced with pretreatment of MSCs with a Toll-like receptor 3 agonist, polyinosinic:polycytidylic acid (Poly (I:C)), prior to the isolation of MVs. Isolated human alveolar macrophages had increased antimicrobial activity with MSC MV treatment in vitro as well. Although oxygenation and lung compliance levels were similar between injury and treatment groups, administration of MSC MVs numerically decreased median pulmonary artery pressure at 6 hours.

Conclusions: In summary, MSC MVs increased alveolar fluid clearance and reduced lung protein permeability, and pretreatment with Poly (I:C) enhanced the antimicrobial activity of MVs in an ex vivo perfused human lung with severe bacteria pneumonia.

Keywords: ards; pneumonia; pulmonary oedema.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1.. Schematic of the Ex Vivo Perfused Human Lung Preparation.

Marginal human lungs which were not used for lung transplantation were obtained from the Northern California Transplantation Donor Network. Using a large organ Harvard bioreactor, the pulmonary artery of the right or left lung was initially cannulated and gently perfused with a crystalloid solution containing 5% albumin at a rate up to 250 ml/min. Once rewarmed, the lung was ventilated using a tidal volume of 300 ml with 5 cmH20 of PEEP and a respiratory rate 10 breaths per minute in room air. AFC rate was first measured in the upper control lobe. If AFC > 0 and <10 %/h, 10 CFU E.coli bacteria was administered into the lower lung lobe, and 100 ml of fresh human blood was added to the perfusate. After 6 h, AFC was measured in the lower lobe as the primary endpoint. In separate experiments and randomly, 200 or 400 μl MSC derived microvesicles (MVs), Poly (I:C) pretreated 200 μl MSC MVs, or 200 μl NHLF derived MVs was administered into the perfusate at 1 h.

Figure 2.. Therapeutic Effect of MSC MVs on Alveolar Fluid Clearance Rate following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

(A) Administration of MSC MVs or Poly (I:C) pretreated MSC MVs intravenously significantly increased the AFC rate in the lung lobe injured by E.coli pneumonia at 6 h. Administration of NHLF MVs had no beneficial effect on AFC rate. AFC was measured by the change in protein concentration of a 5% albumin instillate in the lung lobe over 1 h and expressed as mean AFC (% per h, per 150 ml of BAL fluid) with 95% confidence intervals for each condition. N = 9 for E.coli injured lung lobe, N = 6 for E.coli injured lung lobe treated with MSC MVs (200 μl or 400 μl) or N = 11 for 200 μl MSC MVs pretreated Poly (I:C) or N = 5 for 200 μl NHLF MVs. Overall P Value < 0.001, * p is significant vs. Control, ** p is significant vs. E.coli injured, and *** p is significant vs. NHLF MVs by ANOVA (Bonferroni). P values and confidence intervals for individual pair comparisons are shown in Supplementary Table. (B) Human lungs injured with E.coli bacteria with and without MSC MVs 1X or Poly (I:C) pretreated MSC MVs were fixed in 10% formalin at 6 h. Sections were stained with hematoxylin and eosin. Administration of human MSC MVs 1X or Poly (I:C) pretreated MSC MVs 1 h after E.coli pneumonia injury reduced the level of hemorrhage, edema, and cellularity in the injured lung lobe at 6 h following E.coli pneumonia.

Figure 2.. Therapeutic Effect of MSC MVs on Alveolar Fluid Clearance Rate following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

(A) Administration of MSC MVs or Poly (I:C) pretreated MSC MVs intravenously significantly increased the AFC rate in the lung lobe injured by E.coli pneumonia at 6 h. Administration of NHLF MVs had no beneficial effect on AFC rate. AFC was measured by the change in protein concentration of a 5% albumin instillate in the lung lobe over 1 h and expressed as mean AFC (% per h, per 150 ml of BAL fluid) with 95% confidence intervals for each condition. N = 9 for E.coli injured lung lobe, N = 6 for E.coli injured lung lobe treated with MSC MVs (200 μl or 400 μl) or N = 11 for 200 μl MSC MVs pretreated Poly (I:C) or N = 5 for 200 μl NHLF MVs. Overall P Value < 0.001, * p is significant vs. Control, ** p is significant vs. E.coli injured, and *** p is significant vs. NHLF MVs by ANOVA (Bonferroni). P values and confidence intervals for individual pair comparisons are shown in Supplementary Table. (B) Human lungs injured with E.coli bacteria with and without MSC MVs 1X or Poly (I:C) pretreated MSC MVs were fixed in 10% formalin at 6 h. Sections were stained with hematoxylin and eosin. Administration of human MSC MVs 1X or Poly (I:C) pretreated MSC MVs 1 h after E.coli pneumonia injury reduced the level of hemorrhage, edema, and cellularity in the injured lung lobe at 6 h following E.coli pneumonia.

Figure 3.. Therapeutic Effect of MSC MVs on Bacterial Levels following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of Poly (I:C) pretreated MSC MVs decreased (A) bacterial counts in the BAL fluid at 6 h. Administration of NHLF MVs had no beneficial effect. Data is expressed in the box plot as median bacterial CFU counts with IQR for bars in the BAL fluid, Overall P value = 0.016, ** p is significant vs. E.coli injury group by Post-Hoc Dunn’s Analyses Following Kruskal Wallis Test. (B) Although not statistically significant, administration of MSC MVs or Poly (I:C) pretreated MSC MVs numerically decreased bacterial CFU counts in the perfusate. Data is expressed in the graph as median bacterial CFU counts with IQR for bars in the perfusate. Overall P value = 0.624. (C) Co-incubation of MSC MVs with freshly isolated human alveolar macrophages also increased E.coli bacterial phagocytosis by the macrophages. Data is expressed in the graph as median bacterial CFU counts (as % of LPS control) with IQR for bars. Overall P value = 0.026, * p is significant vs. LPS treated group by Post-Hoc Dunn’s Analyses Following Kruskal Wallis test. P values and confidence intervals for individual pair comparisons are shown in Supplementary Table.

Figure 3.. Therapeutic Effect of MSC MVs on Bacterial Levels following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of Poly (I:C) pretreated MSC MVs decreased (A) bacterial counts in the BAL fluid at 6 h. Administration of NHLF MVs had no beneficial effect. Data is expressed in the box plot as median bacterial CFU counts with IQR for bars in the BAL fluid, Overall P value = 0.016, ** p is significant vs. E.coli injury group by Post-Hoc Dunn’s Analyses Following Kruskal Wallis Test. (B) Although not statistically significant, administration of MSC MVs or Poly (I:C) pretreated MSC MVs numerically decreased bacterial CFU counts in the perfusate. Data is expressed in the graph as median bacterial CFU counts with IQR for bars in the perfusate. Overall P value = 0.624. (C) Co-incubation of MSC MVs with freshly isolated human alveolar macrophages also increased E.coli bacterial phagocytosis by the macrophages. Data is expressed in the graph as median bacterial CFU counts (as % of LPS control) with IQR for bars. Overall P value = 0.026, * p is significant vs. LPS treated group by Post-Hoc Dunn’s Analyses Following Kruskal Wallis test. P values and confidence intervals for individual pair comparisons are shown in Supplementary Table.

Figure 3.. Therapeutic Effect of MSC MVs on Bacterial Levels following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of Poly (I:C) pretreated MSC MVs decreased (A) bacterial counts in the BAL fluid at 6 h. Administration of NHLF MVs had no beneficial effect. Data is expressed in the box plot as median bacterial CFU counts with IQR for bars in the BAL fluid, Overall P value = 0.016, ** p is significant vs. E.coli injury group by Post-Hoc Dunn’s Analyses Following Kruskal Wallis Test. (B) Although not statistically significant, administration of MSC MVs or Poly (I:C) pretreated MSC MVs numerically decreased bacterial CFU counts in the perfusate. Data is expressed in the graph as median bacterial CFU counts with IQR for bars in the perfusate. Overall P value = 0.624. (C) Co-incubation of MSC MVs with freshly isolated human alveolar macrophages also increased E.coli bacterial phagocytosis by the macrophages. Data is expressed in the graph as median bacterial CFU counts (as % of LPS control) with IQR for bars. Overall P value = 0.026, * p is significant vs. LPS treated group by Post-Hoc Dunn’s Analyses Following Kruskal Wallis test. P values and confidence intervals for individual pair comparisons are shown in Supplementary Table.

Figure 4.. Effect of MSC MVs on the Influx of Inflammatory Cells following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Instillation of MSC MVs numerically decreased the influx of inflammatory cells, specifically neutrophils, into the lung lobe injured by E.coli pneumonia at 6 h. Absolute neutrophil count is expressed as median with IQR for bars for each condition, N = 9 for E.coli injured lung lobe, N = 6 – 11 for E.coli–injured lung lobe treated with 200 μl or 400 μl of MSC MVs or 200 μl of Poly (I:C) pretreated MSC MVs. Overall P value = 0.024, P values and confidence intervals for individual pair comparisons are shown in Supplementary Table.

Figure 5.. Therapeutic Effect of MSC MVs on Lung Protein Permeability following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Lung protein permeability at 6 h as measured by Evan’s Blue extravasation. (A) Administration of Evan’s Blue intravenously demonstrated albumin protein leak into the E.coli bacteria injured lung lobe. (B) Compared to E.coli bacteria injured lung lobe, administration of MSC MVs or Poly (I:C) pretreated MSC MVs significantly reduced lung protein permeability. Data is presented as mean change in permeability with 95% confidence intervals. Overall P value = 0.001, * P is significant vs. E.coli by ANOVA (Bonferroni). P values and confidence intervals for individual pair comparisons are shown in Supplementary Table.

Figure 6.. Effect of MSC MVs on Pulmonary Arterial Pressure and Vascular Resistance following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of MSC MVs or Poly (I:C) pretreated MSC MVs numerically decreased PAP (A) or PVR (B) at T6 h compared to E.coli injury group. Data is presented as median PAP or PVR with IQR as bars. P = 0.20 – 0.66 for each time point by Kruskal Wallis tests (A). P = 0.20 – 0.63 for each time point by Kruskal Wallis tests (B).

Figure 6.. Effect of MSC MVs on Pulmonary Arterial Pressure and Vascular Resistance following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of MSC MVs or Poly (I:C) pretreated MSC MVs numerically decreased PAP (A) or PVR (B) at T6 h compared to E.coli injury group. Data is presented as median PAP or PVR with IQR as bars. P = 0.20 – 0.66 for each time point by Kruskal Wallis tests (A). P = 0.20 – 0.63 for each time point by Kruskal Wallis tests (B).

Figure 7.. Lack of a Therapeutic Effect of MSC MVs on Lung Compliance or Oxygenation following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of MSC MVs or Poly (I:C) pretreated MSC MVs had no significant effect on tracheal pressure (A), lung compliance (B) or oxygenation (C) over 6 h. Data is presented as median Tracheal pressure or Lung Compliance or PaO2 with IQR as bars. P = 0.56 – 0.98 for each time point by Kruskal Wallis tests (A). P = 0.56 – 0.98 for each time point by Kruskal Wallis tests (B). P = 0.10 – 0.92 for each time point by Kruskal Wallis tests (C).

Figure 7.. Lack of a Therapeutic Effect of MSC MVs on Lung Compliance or Oxygenation following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of MSC MVs or Poly (I:C) pretreated MSC MVs had no significant effect on tracheal pressure (A), lung compliance (B) or oxygenation (C) over 6 h. Data is presented as median Tracheal pressure or Lung Compliance or PaO2 with IQR as bars. P = 0.56 – 0.98 for each time point by Kruskal Wallis tests (A). P = 0.56 – 0.98 for each time point by Kruskal Wallis tests (B). P = 0.10 – 0.92 for each time point by Kruskal Wallis tests (C).

Figure 7.. Lack of a Therapeutic Effect of MSC MVs on Lung Compliance or Oxygenation following Severe Escherichia coli Pneumonia in Ex Vivo Perfused Human Lung.

Administration of MSC MVs or Poly (I:C) pretreated MSC MVs had no significant effect on tracheal pressure (A), lung compliance (B) or oxygenation (C) over 6 h. Data is presented as median Tracheal pressure or Lung Compliance or PaO2 with IQR as bars. P = 0.56 – 0.98 for each time point by Kruskal Wallis tests (A). P = 0.56 – 0.98 for each time point by Kruskal Wallis tests (B). P = 0.10 – 0.92 for each time point by Kruskal Wallis tests (C).